Seminar 2

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Immuno histo chemistry : 

Immuno histo chemistry Prepared by: Dr.Sourab Kumar

contents : 

contents Definition Historical background Basic concepts of immunohistochemistry Troubleshooting in immunohistochemistry Marker Proteins used in immunohistochemistry

Definitions : 

Definitions Immunohistochemistry is a technique for identifying tissue constituents (antigens) by means of antigen-antibody interactions. The site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary antibody labeling method.

A.H.Coons : 

A.H.Coons In 1940, (Microbiologist) described identification of tissue antigens in pneumococci by using direct fluorescence

Applications : 

Applications Cancer diagnostics Differential diagnosis Treatment of cancer Research

Alteration of tissue architecture : 

Alteration of tissue architecture

Slide 7: 

IMMUNOHISTOCHEMISTRY STEPS

Tissue sections : 

Tissue sections

TISSUE SECTIONS : 

TISSUE SECTIONS Fresh tissue biopsy sections Old Biopsy sections(Registries, Old paraffin blocks) Fresh biopsies with frozen techniques Cytology smears Adhesives

Slide 11: 

After tissue sectioning let us start with IHC procedure, that is antigen retrieval.

Slide 12: 

But before that, we have to carry out two steps prior to undertaking the antigen retrieval procedure

Blocking Endogenous Enzymes : 

Blocking Endogenous Enzymes Why???

Blocking endogenous enzymes : 

Blocking endogenous enzymes 10min

Biocare products available for blocking endogenous enzymes : 

Biocare products available for blocking endogenous enzymes

Buffer solutions : 

Buffer solutions Wash the slide thoroughly with TRIS neutralized buffered solutions 30 Sec

Slide 17: 


Buffer Solutions : 

Buffer Solutions

Slide 19: 

Now shall we proceed with antigen retrieval procedure??? ?

Slide 20: 

NO

Blocking background staining : 

Blocking background staining Why???

Blocking background staining : 

Blocking background staining Specific. 15min Immunoglobulins and fibrinogens Non-specific. Previous infections of animal Inflammatory exudate/Necrosis Manufacturer

Slide 23: 

Background Staining

Buffer solutions : 

Buffer solutions Wash the slide thoroughly with TRIS neutralized buffered solutions 30 Sec

Slide 25: 

What is antigen Retrieval(Bring back the antigens)? Where have they gone?

Antigen Retrieval Purpose : 

Antigen Retrieval Purpose Epitopes or antigen binding sites are not clear. Breaks the formalin induced cross-linking bonds between epitopes and unrelated proteins, thereby allowing better penetration of antibody and accessibility of epitopes.

Advantages of antigen retrieval : 

Advantages of antigen retrieval Enhanced exposure of antigenic epitopes on surface of tissue section. Allows for reduction in incubation time of primary antibodies or increase dilution of primary antibodies. Consistent and reliable staining. Reduces false negatives in formalin-fixed paraffin embedded tissue sections. Ease of use.

Procedure of antigen retrieval : 

Procedure of antigen retrieval The retrieval techniques of unmasked antigens : (1) proteolytic enzyme digestion (2) microwave (3) microwave and trypsin (4) pressure cooker

Proteolytic enzyme digestion : 

Proteolytic enzyme digestion pH 7.8; 0.1MNaOH; 37degree C

Slide 31: 

Multiple chamber couplin Jar 750W for 10min

Slide 32: 

Microwave Oven 10 min

Slide 34: 

Pressure Cooker 3 Liters ; pH 6.0; 15psi 2min

Distributors in india : 

Distributors in india Allied Scientific Products39/19B Gopal Nagar RoadKolkata-700027India Website http://www.alliedscientific.net Freephone 1800 345 7777 Fax +91 33 24566683 / 24790626 Tel +91 33 24566684/24793109/9039 Contact us Webmaster: webmaster@abcam.com Careers: careers@abcam.com Licensing: licensing@abcam.com Reference: G:\Immunohistochemistry\kolkata distributor.htm

Slide 36: 

Steamer 40min

Antibody treatment : 

Antibody treatment Direct indirect

Direct Method : 

Direct Method Tissue Antigen Labeled Antibody 30min

Two-Step Indirect Method(Most widely used) : 

Two-Step Indirect Method(Most widely used) Tissue Antigen Primary Antibody Secondary Antibody

Types of indirect technique : 

Types of indirect technique ABC method PAP and APAAP method Dextran polymer conjugated two-step visualization system Immunogold silver staining technique Hapten labelling technique Mirror image complementary antibody labelling technique

ABC Method (avidin-biotin complex method ) : 

ABC Method (avidin-biotin complex method )

Slide 55: 

Enzyme and chromogen substrate added

PAP Method (peroxidase anti-peroxidase method) : 

PAP Method (peroxidase anti-peroxidase method)

Slide 57: 

IGSS technique

SP Method(streptavidin peroxidase conjugated method) : 

SP Method(streptavidin peroxidase conjugated method)

Labels : 

Labels

Slide 60: 

DAB Solutions

Chromogens : 

Chromogens Antigen+Antibody 2nd Antibody+Chromogen(HRP) Why we have to add chromogens??? What is the necessity???

IHC automatic stainer : 

IHC automatic stainer

Slide 71: 

IMMUNOHISTOCHEMISTRY STEPS

Troubleshooting : 

Troubleshooting 1. Weak or No Staining 2. Over-staining 3. High Background

High Background : 

High Background

Over-staining : 

Over-staining

Weak or No Staining : 

Weak or No Staining

Weak or No Staining : 

Weak or No Staining

ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS : 

ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS KERATIN S100 EMA HMB45 CD45 MART-1 CD15 FLI-1 VIMENTIN SYN DES CD56 MSA CD99 MYOGENIN

ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS : 

ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS KERATIN S100 VIMENTIN HMB45 BER-EP4 MART-1 CD45 CEA CD15 THROMBOMODULIN MOC31 CALRETININ CD117

REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS : 

REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS KERATIN S100 PROTEIN EMA CD56 VIMENTIN CD31 DESMIN CD34 MSA CD57 THROMBOMODULIN CD99

REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS : 

REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS KERATIN HMB45 EMA MART-1 VIMENTIN THROMBOMODULIN DESMIN CD31 MSA CD34 S100 PROTEIN CD99

REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS : 

REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS CD15 CD79A CD20 LYSOZYME CD30 MYELOPEROXIDASE CD43 CD3 CD45 CD5 CD68 CD163

Slide 82: 

Epithelium: Keratins --pan-keratin and antibodies to keratins of different molecular weights Supporting connective tissues: --Vimentin--fibroblasts, blood vessels --vWF, CD31 (PECAM)-- endothelial cells of blood vessels Hematopoeitic tissues: CD45, B220, CD3, F480, Mac-1, Gr-1, CD41 Muscle: desmin, smooth muscle actin Neural: GFAP, NeuN, F480/Mac-1, MBP, NSE, S100 Hormones: specific antibodies--insulin, casein, etc. Germ cells: alpha-feto protein (teratomas) Proliferation markers-Ki-67

Cytokeratins : 

Cytokeratins Epithelial marker protein

HMB 45 : 

HMB 45

S-100 : 

S-100 .

P 63 : 

P 63

Ki 67 : 

Ki 67

High-grade fibrosarcoma. Spindle-shaped lesional cells have blunt-ended nuclei and here are surrounded by minimal fibrous stroma. Many mitotic figures are seen(p53) : 

High-grade fibrosarcoma. Spindle-shaped lesional cells have blunt-ended nuclei and here are surrounded by minimal fibrous stroma. Many mitotic figures are seen(p53)

HPV : 

HPV

References : 

References Bancroft histological procedures Ihc world.co.in Culling histological procedures4. Journals of Oral Pathology and Medicine 5. Shafer’s oral pathology

Thank you ! : 

Thank you !

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